This project aims to understand the cytokine control of megakaryocytes and platelet production, with the long-term goal of devising better clinical strategies for the management of thrombocytopenias associated with cancer treatment and other disorders. The recent cloning of TPO has provided a molecule with potent capacity to stimulate megakaryocyte and platelet generation. The thrombocytopenia in mice lacking TPO or its receptor c-Mpl is evidence that TPO is the dominant physiologic regulator of megakaryocytopoiesis, but that sufficient TPO-independent production occurs to maintain hemostasis. Defining the cytokine control of the TPO-independent processes and the mechanisms by which TPO interacts with other regulators is a key step in determining the most effective clinical application of TPO and other megakaryocytopoietic agents. The current proposal aims to define the roles of known regulators of megakaryocytopoiesis: IL-3, IL-6, IL-11, GM-CSF, LIF, and SCF, in residual steady-state as well as emergency megakaryocyte and platelet production in Mpl-deficient mice, although analysis of compound mutant mice lacking these regulators or their receptors in addition of c-Mpl. Preliminary results suggest that TPO signaling also plays a critical role in regulation of the hemopoietic stem cell compartment. If TPO is to be administered to patients, particularly those with leukemia, its actions on stem cells must be fully understood. Analysis of specific stem cell populations, isolated by cell sorting strategies, in long term hemopoietic reconstitution assays will be used to precisely define the stem cell deficiency in Mpl-deficient mice and in compound mutants lacking c-Mpl and other stem cell regulators (IL-6, LIF, Il-11, SCF, G-CSF). Stem cells from normal mice that express the c-Mpl receptor will also be isolated and functionally characterized by generating knock-in mice expressing green fluorescent protein under the control of the c-mpl locus. An understanding of the specific biological responses stimulated by signals from different regions of the c-Mpl receptor may ultimately allow identification of intracellular targets for therapeutic manipulations of the TPO responses. Incorporation of c-Mpl cytoplasmic domain mutations into the mouse germline will be used to determine if the distinct cytoplasmic regions of the TPO receptor implicated in proliferation and differentiation in vitro, also mediate these responses in the physiological regulation of megakaryocytopoiesis and stem cells.